CN110129411A - The active detection method of Glucosamine synthase expression in a kind of Escherichia coli - Google Patents
The active detection method of Glucosamine synthase expression in a kind of Escherichia coli Download PDFInfo
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Abstract
The present invention relates to the active detection methods of Glucosamine synthase expression in a kind of Escherichia coli.The present invention is for the first time using the method for detecting GlmS enzyme activity in Bacillus coli cells based on the generating rate of enzyme reaction product glutamic acid, it passes through special pre-treatment step, condition of culture and breaking-wall cell condition to Escherichia coli are optimized, guarantee the synchronism of detection process and the comparativity of testing result, detection accuracy is significantly improved, to guarantee that the testing result tool of the detection method has significant practical applications；And this method is easy to operate, it is easy to control, detection efficiency can be improved, bio-sensing analysis-e/or determining content of glutamic acid can effectively be utilized, method to quickly measure Glucosamine synthase activity, while also greatly facilitating the research to GlmS structure-function relationship.
Description
Technical field
The present invention relates to the active detection methods of Glucosamine synthase expression in a kind of Escherichia coli, belong to microbial enzyme
Technical field.
Background technique
Glucosamine synthase (GlmS) can be catalyzed glutamine (Glutamine) and fructose-6-phosphate (Fru-6-P)
Reaction, with the hydrolysis of Glutamine, amino is shifted and is completed the isomerization of amino sugar, and GlmS successively releases paddy ammonia
Acid and 6- phosphorylated amino glucose (GlcN-6-P), the reaction are synthesizing amino hexoses and then complete whole cell peptidoglycan synthesis
First step reaction, the final product UDP-N- acetylglucosamine of the route of synthesis is bacterium and fungal cell wall basis
Constituent.In bacteria cell wall, the basis of the big main macromolecular of peptide glycan, lipopolysaccharides two is acetylglucosamine
(GlcNAc) and Glucosamine (GlcN).Glucosamine synthase (GlmS) is the key enzyme for synthesizing GlcNAc and GlcN, because
This enzyme is of great significance to cells survival.Feedback inhibition of the enzyme by its metabolite GlcN-6-P.
As the direct product of GlmS, it is a kind of presence that Glucosamine (Glucosamine, GlcN), which is also known as ammonia sugar,
The chief component of amino monosaccharide or glycoprotein in the structures such as connective tissue.Glucosamine is closed in human synovial
It also plays an important role in proteoglycans.The purposes of aminoglucose carbohydrates and their derivative is very extensive, especially in food,
Medicine and other fields.
It is the trend of the following Glucosamine industry development, this method with microbial method synthetic method production Glucosamine
Raw material is sufficient, working condition is mild, the sensitizer for not generating strong acid and strong base waste water, being free of allergic constitution crowd in product.Greatly
Enterobacteria has the advantages such as growth cycle is short, hereditary information understands, molecular biology manipulations facilitate, and becomes bioanalysis synthesizing amino
The most common host strain of glucose, the principle of this method be by molecular biology method, after the GlmS of object bacteria is cloned,
It is integrated on the plasmid or chromosome of Escherichia coli, and the inducing expression in Escherichia coli, synthesizes GlmS in the cell, and secrete
Into fermentation liquid.Since the source of GlmS is different, the construction strategy of engineering bacteria is different, expression efficiency of the GlmS in Escherichia coli
Difference, causes the production ammonia sugar performance difference of Glucosamine synthesis bacterium very big or even same strain bacterium is under same culture conditions
Production ammonia sugar level there is also biggish fluctuatings.This shows that GlmS in expression effect intracellular is bioanalysis synthesizing amino glucose
Key problem, and the enzyme in expression intracellular at present there is no intuitive detection method, cause the transformation to thallus to be imitated
Fruit cannot accurately be timely feedbacked from molecular level, meanwhile, it is also no efficient, quick to the big flux screening of Producing Strain
Method.The existing evaluation method to Glucosamine synzyme is after strain fermentation end cycle, and measurement reaction generates
The yield of Glucosamine is screened, and that there are thallus cultivation cycles is long for this method, operating procedure is more, cannot reflect thallus culture
In the process the problems such as GlmS enzyme activity situation of change.And the detection of Glucosamine synthase can not also use for reference other enzyme activity of this field
Detection method, such as:
Chinese patent literature CN104101595A (application number 201310132760.3) discloses a kind of using colorimetric method inspection
Survey the detection method of farnesyl pyrophosphate synthase, the product pyrophosphoric acid or phosphorus that this method uses colorimetric determination wherein to generate
Acid, then reacted with it with particular agent, coloured compound, its concentration of colorimetric determination are formed after reduction.But this method makes
Colorimetric method step is complicated, accuracy is not high, can not accurate detection farnesyl pyrophosphate synthase activity.
Chinese patent literature CN102747055A (application number 201210204587.9) discloses a kind of extraction pear fruit sugarcane
Sugared synthase and Sucrose Phosphate Synthase and its activity determination method, this method increases in synzyme extraction process saltouts and dialyses
Step, to measure the activity of sucrose synthase and Sucrose Phosphate Synthase in fruit.But this method, which needs to increase, saltouts and dialyses
Step, process is complicated, accuracy is not high, can not quick and precisely detect the activity of sucrose synthase and Sucrose Phosphate Synthase.
But since Glucosamine synthase is one of the key enzyme for influencing Bacillus coli cells wall synthesis main component, because
This, its product is directly detected using the above method, will receive the influence of preprocessing process, extreme influence testing result it is accurate
Property, this is also why active to evaluate Glucosamine synthase expression without directly detection Glucosamine synthase product at present
Reason.
Summary of the invention
The present invention is directed to the deficiency of existing technology, provides the active detection of Glucosamine synthase expression in a kind of Escherichia coli
Method.
GlmS catalyst mechanism is as follows:
Technical solution of the present invention is as follows:
The active detection method of Glucosamine synthase expression in a kind of Escherichia coli, steps are as follows:
(1) the recombination bacillus coli sample to be tested of the plasmid containing pET28a-glmS is taken, it is activated, activated strains are made；
(2) activated strains obtained in step (1) are inoculated in the fluid nutrient medium containing kanamycins, 30~40
DEG C, 180~220r/min shaking table culture to OD600When reaching 3~8, addition inducer IPTG to concentration is 1~2.5mmol/L,
In 20~35 DEG C, 180~220r/min shaking table 8~12h of Fiber differentiation, it is made and gives full expression to bacterial strain；
(3) bacterial strain will be given full expression to made from step (2) to be separated by solid-liquid separation, cleans thallus, is then resuspended, be made and bacterium is resuspended
Body；Then thallus will be resuspended and be placed in 25~35min of freezing in -80 DEG C of low temperature environments below, be subsequently placed in 35~38 DEG C of water-baths
25~35min of middle defrosting, repeated freezing, defrosting step 2~4 time；Then it is centrifuged, collects supernatant, GlmS crude enzyme liquid is made；
(4) at room temperature, after taking quantitative substrate solution to mix with GlmS crude enzyme liquid made from quantitative step (3),
30~38 DEG C of 15~30min of reaction terminate reaction, and centrifugation takes supernatant, detects glutamic acid yield, calculate obtain table according to the following formula
Up to activity:
In formula: VEnzyme: react the enzyme solution volume (μ l) of addition
CGlutamic acid: aminoglutaric acid concentration (mg/dl)
VAlways: the final volume (μ l) of Glucosamine synthase enzyme activity determination reaction system
T: reaction time (min)
147: glutamate molecule quality.
It is preferred according to the present invention, in the step (1), activate as in 35~38 DEG C, 180~220r/min shaking table culture
To OD600Reach 5.5~6.0.
It is preferred according to the present invention, in the step (1), the culture medium used is activated as containing 50 μ g/mL kanamycins
LB liquid medium.
Preferred according to the present invention, in the step (2), the fluid nutrient medium containing kanamycins is containing 50 μ g/mL of concentration
The LB liquid medium of kanamycins.
The LB liquid medium is this field conventional medium, is usually used in the culture and induction of Escherichia coli, component is such as
Under:
Peptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
It is preferred according to the present invention, it in the step (3), is separated by solid-liquid separation to be centrifuged, condition is 8000r/min centrifugation
5min。
It is preferred according to the present invention, in the step (3), cleans thallus and resuspension is all made of concentration 0.1mol/L PBS and delays
Fliud flushing is cleaned 2~3 times.
It is preferred according to the present invention, in the step (3), it is centrifuged to be centrifuged 20min under the conditions of 12000r/min.
Preferred according to the present invention, in the step (4), substrate solution component is as follows: 2~5mg/L glutamine, 5~
8mg/L fructose-1, 6-diphosphate, 0.7~1mg/L ethylenediamine tetra-acetic acid, the 100mmol/L phosphate buffer that 1mL pH value is 7.5.
Preferred according to the present invention, in the step (4), the volume ratio of substrate solution and GlmS crude enzyme liquid is 5:1.
Preferred according to the present invention, in the step (4), termination reaction condition is 95 DEG C of processing 4min.
Preferred according to the present invention, in the step (4), centrifugal condition is that 8000r/min is centrifuged 5min.
Preferred according to the present invention, in the step (4), detection glutamic acid yield is to be examined using biosensor
It surveys.
Beneficial effect
The present invention is used detected GlmS in Bacillus coli cells based on the generating rate of enzyme reaction product glutamic acid for the first time
The method of enzyme activity, by special pre-treatment step, by condition of culture to Escherichia coli and breaking-wall cell condition into
It has gone optimization, has guaranteed the synchronism of detection process and the comparativity of testing result, significantly improve detection accuracy, so that guaranteeing should
The testing result tool of detection method has significant practical applications；And this method is easy to operate, it is easy to control, detection effect can be improved
Rate can effectively utilize bio-sensing analysis-e/or determining content of glutamic acid, to quickly measure Glucosamine synthase activity
Method, while also greatly facilitating the research to GlmS structure-function relationship.
Detailed description of the invention
601L point saturation mutation strain enzyme activity distribution map in Fig. 1 GlmS；
602A point saturation mutation strain enzyme activity distribution map in Fig. 2 GlmS；
Fig. 3 genetic engineering bacterium AG33 (BL21-pETDute-1-glmS-gna1), AGM56 (BL21-pETDuet-1-
GlmS-gna △ manX), control bacterium (BL21) aminoglutaric acid concentration compare histogram；
Fig. 4 genetic engineering bacterium AG33 (BL21-pETDute-1-glmS-gna1), AGM56 (BL21-pETDuet-1-
GlmS-gna △ manX), control bacterium (BL21) enzyme activity compare histogram；
Specific embodiment
Technical solution of the present invention is further elaborated below with reference to embodiment, but the range that the present invention is protected is unlimited
In this.
The definition of Glucosamine synthase enzyme activity: at 37 DEG C of optimum condition, 1 μm of ol glutamine conversion of catalysis per minute
A unit of activity, i.e. 1U are defined as enzyme amount required for glutamic acid.
VEnzyme: react the enzyme solution volume (μ l) of addition
CGlutamic acid: aminoglutaric acid concentration (mg/dl)
VAlways: the final volume (μ l) of Glucosamine synthase enzyme activity determination reaction system
T: reaction time (min)
147: glutamate molecule quality
Sample source
Clonal expression host strain is E.coli BL21 (DE3) competent cell, is purchased from Vazyme company；AG33
(pETDuet-1-glmS-gna1), AGM56 (pETDuet-1-glmS-gna △ manX), pET28a-glmS plasmid are referring to (king
Rise building and recombinase zymology Quality Research [D] .2015. of Glucosamine synthase gene engineering bacteria) record in document
It is constructed, set out plasmid AG33, AGM56, pET28a are carrier commonly used in the art, can be bought from market, pETDuet-1
Plasmid is purchased from Vazyme company.
Embodiment 1
The active detection method of Glucosamine synthase expression in a kind of Escherichia coli, steps are as follows:
(1) this laboratory is taken to carry out 96 plant mutant bacterial strains of rite-directed mutagenesis to the 601st L in GlmS nucleic acid sequence
(E.coliBL21 plasmid containing pET28a-glmS) is inoculated in respectively in 1mL LB liquid medium (containing 50 μ g/mL kanamycins),
37 DEG C are put into, 200r/min shaking table is incubated overnight to OD600Reach 6.0, activated strains are made；
(2) by 96 plants of activated strains obtained in step (1) be inoculated in 1mL LB liquid medium (containing 50 μ g/mL cards that
Mycin) in, 40 DEG C are put into, 200r/min shaking table culture to OD600When reaching 3, inducer IPTG to final concentration 2.5mmol/ is added
L is made at 20 DEG C, 200r/min shaking table culture 12h and gives full expression to bacterial strain；
(3) the bacterium solution 8000r/min centrifugation 5min that step (2) induction terminates the thallus that induction terminates is collected to use
0.1mol/LPBS cleaning thallus removes remaining culture medium twice, and 100 μ L 0.1mol/L PBS suspension cell again is added；Carefully
Born of the same parents freeze repeatedly can make cell rupture due to foring increasing for salinity in ice crystal and remaining liq in cell with when melting,
Again the cell to suspend is put into -80 DEG C of refrigerators and is rapidly frozen 30min, then is put into 37 DEG C of water-baths the 30min that thaws rapidly, weight
Again three times；12000r/min is centrifuged 20min and collects supernatant after clasmatosis, and GlmS crude enzyme liquid is made；
(4) it takes 100 μ L substrates to be added in 200 μ L EP pipes, then 20 μ L enzyme solutions is taken to be added on EP tube wall, to all add
Afterwards, while centrifugation makes substrate be sufficiently mixed to ensure to react with enzyme solution and start simultaneously at, and is put on 38 DEG C of insulation boards and reacts 15min,
4min is handled at 95 DEG C and terminates reaction, and centrifuging and taking supernatant detects glutamic acid yield using SBA, filters out the higher bacterial strain of enzyme activity；
Detection related data is as follows respectively:
VEnzyme: react the enzyme solution volume 20 (μ l) of addition
CGlutamic acid: aminoglutaric acid concentration (mg/dl)
VAlways: the final volume 120 (μ l) of Glucosamine synthase enzyme activity determination reaction system
T: reaction time 30 (min)
Through detecting, related test results are as shown in table 1:
1 catastrophe point L-Glu Concentration Testing result of table
Following formula is substituted into according to above-mentioned data:
It is computed, as a result as shown in Figure 1.
2 mutant bacteria A enzyme activity of embodiment improves bacterial strain screening
The active detection method of Glucosamine synthase expression in a kind of Escherichia coli, steps are as follows:
(1) this laboratory is taken to carry out 96 plant mutant bacterial strains of rite-directed mutagenesis to the 602nd A in GlmS nucleic acid sequence
(E.coliBL21 plasmid containing pET28a-glmS) is inoculated in respectively in 1mL LB liquid medium (containing 50 μ g/mL kanamycins),
37 DEG C are put into, 200r/min shaking table is incubated overnight to OD600Reach 5.5, activated strains are made；
(2) by 96 plants of activated strains obtained in step (1) be inoculated in 1mL LB liquid medium (containing 50 μ g/mL cards that
Mycin) in, 40 DEG C are put into, 200r/min shaking table culture to OD600When reaching 8, inducer IPTG to final concentration 1mmol/L is added,
35 DEG C, 200r/min shaking table culture 8h are put into, is made and gives full expression to bacterial strain；
(3) the bacterium solution 8000r/min centrifugation 5min that step (2) induction terminates the thallus that induction terminates is collected to use
0.1mol/LPBS cleaning thallus removes remaining culture medium twice, and 100 μ L 0.1mol/L PBS suspension cell again is added；Carefully
Born of the same parents freeze repeatedly can make cell rupture due to foring increasing for salinity in ice crystal and remaining liq in cell with when melting,
Again the cell to suspend is put into -80 DEG C of refrigerators and is rapidly frozen 30min, then is put into 37 DEG C of water-baths the 30min that thaws rapidly, weight
Again three times；12000r/min is centrifuged 20min and collects supernatant after clasmatosis, and GlmS crude enzyme liquid is made；
(4) it takes 100 μ L substrates to be added in 200 μ L EP pipes, then 20 μ L enzyme solutions is taken to be added on EP tube wall.To all add
Afterwards, while centrifugation makes substrate be sufficiently mixed to ensure to react with enzyme solution and start simultaneously at.It is put on 30 DEG C of insulation boards and reacts 30min,
4min is handled at 95 DEG C and terminates reaction, and centrifuging and taking supernatant detects glutamic acid yield using SBA, filters out the higher bacterial strain of enzyme activity；
Detection related data is as follows respectively:
VEnzyme: react the enzyme solution volume 20 (μ l) of addition
CGlutamic acid: aminoglutaric acid concentration (mg/dl)
VAlways: the final volume 120 (μ l) of Glucosamine synthase enzyme activity determination reaction system
T: reaction time 30 (min)
Through detecting, related test results are as shown in table 2:
2 catastrophe point A aminoglutaric acid concentration testing result of table
Following formula is substituted into according to above-mentioned data:
It is computed, as a result as shown in Figure 2.
Embodiment 3
Respectively to genetic engineering bacterium AG33 (BL21-pETDute-1-glmS-gna1), the AGM56 of laboratory transformation
(BL21-pETDuet-1-glmS-gna △ manX), control bacterium (BL21) carry out the analysis of GlmS enzyme activity, and obtain related enzyme activity
The reduced parameter of power；
Detection related data is as follows respectively:
VEnzyme: react the enzyme solution volume 20 (μ l) of addition
CGlutamic acid: aminoglutaric acid concentration (mg/dl)
VAlways: the final volume 120 (μ l) of Glucosamine synthase enzyme activity determination reaction system
T: reaction time 30 (min)
Through detecting, related test results are as shown in Figure 3:
Following formula is substituted into according to above-mentioned data:
It is computed, as a result as shown in Figure 4.
Comparative example
Method as described in Example 1, the difference is that, enzyme activity determination method is to use after the completion of reaction
Elson-Morgan method detects the amount of the Glucosamine (GlcN) generated in fermentation liquid to carry out.Elson-Morgan
Specific step is as follows by method detection GlcN: taking 0.5mL sample supernatant that 1.0mL acetylacetone,2,4-pentanedione reagent, 90 DEG C of water bath processings are added
1h is cooled to room temperature, and is slowly added into the dehydrated alcohol of 10mL96% (v/v), not stirred bottom liquid and (be had apparent layering
Phenomenon), add the DMAB reagent of 1.0mL, be uniformly mixed, be put into 25 DEG C of water-bath, mildly react 1h, at 530nm into
Row colorimetric calculates the content of GlcN according to standard curve.
Enzyme-activity unit is defined as at 37 DEG C, and enzyme amount needed for reaction generates 1 μm of olGlcN per minute is defined as an enzyme activity
Unit.
The entire detection process of Elson-Morgan method does not directly contact GlmS enzyme intracellular, moreover, GlcN is intracellular
Phosphorylated amino glucose (GlcNP) is first synthesized under the action of GlmS enzyme, it is extracellular using can be just secreted into after transhipment dephosphorylation,
Period needs multiple reactions and transfer step, has certain lag, accuracy decline with the activity of data instruction GlmS enzyme.
Since Elson-Morgan method is detected as indirectly measuring the active method of GlmS enzyme, side of the invention
Method is easy to operate, it is easy to control, can reflect that enzyme activity changes in real time, be measurement foundation with GlmS enzyme reaction direct product, data are quasi-
True property is high, can utilize bio-sensing analyzer batch measurement content of glutamic acid, additionally it is possible to which the collimation for improving multi-group data is one
Accurate, the reliable GlmS enzyme assay method of kind.Two kinds of GlmS Enzyme activity assay methods detect same sample, and the results are shown in Table 4.
4 two kinds of GlmS Enzyme activity assay method comparing results of table
Note: method 1 detects GlmS enzyme activity in Bacillus coli cells based on the generating rate of enzyme reaction product glutamic acid
Method
The method that method 2 detects GlmS enzyme activity in Bacillus coli cells based on the generating rate of enzyme reaction product GlcN
Claims (10)
1. the active detection method of Glucosamine synthase expression in a kind of Escherichia coli, which is characterized in that steps are as follows:
(1) the recombination bacillus coli sample to be tested of the plasmid containing pET28a-glmS is taken, it is activated, activated strains are made；
(2) activated strains obtained in step (1) are inoculated in the fluid nutrient medium containing kanamycins, in 30~40 DEG C, 180
~220r/min shaking table culture is to OD600When reaching 3~8, addition inducer IPTG to concentration is 1~2.5mmol/L, 20~
35 DEG C, 180~220r/min shaking table 8~12h of Fiber differentiation are made and give full expression to bacterial strain；
(3) bacterial strain will be given full expression to made from step (2) to be separated by solid-liquid separation, cleans thallus, is then resuspended, be made and thallus is resuspended；
Then thallus will be resuspended and be placed in 25~35min of freezing in -80 DEG C of low temperature environments below, be subsequently placed in 35~38 DEG C of water-baths and solve
Freeze 25~35min, repeated freezing, defrosting step 2~4 time；Then it is centrifuged, collects supernatant, GlmS crude enzyme liquid is made；
(4) at room temperature, after taking quantitative substrate solution to mix with GlmS crude enzyme liquid made from quantitative step (3), 30~
38 DEG C of 15~30min of reaction terminate reaction, and centrifugation takes supernatant, detects glutamic acid yield, and calculating obtains expression and lives according to the following formula
Property:
In formula: VEnzyme: react the enzyme solution volume (μ l) of addition
CGlutamic acid: aminoglutaric acid concentration (mg/dl)
VAlways: the final volume (μ l) of Glucosamine synthase enzyme activity determination reaction system
T: reaction time (min)
147: glutamate molecule quality.
2. detection method as described in claim 1, which is characterized in that in the step (1), activate as in 35~38 DEG C, 180
~220r/min shaking table culture is to OD600Reach 5.5~6.0；
Preferably, in the step (1), the culture medium used is activated as the LB liquid medium containing 50 μ g/mL kanamycins.
3. detection method as described in claim 1, which is characterized in that in the step (2), the Liquid Culture containing kanamycins
Base is the LB liquid medium of the 50 μ g/mL kanamycins containing concentration.
4. detection method as described in claim 1, which is characterized in that in the step (3), be separated by solid-liquid separation as centrifugation, condition
5min is centrifuged for 8000r/min.
5. detection method as described in claim 1, which is characterized in that in the step (3), clean thallus and resuspension is all made of
Concentration 0.1mol/L PBS buffer solution is cleaned 2~3 times；
Preferably, it in the step (3), is centrifuged to be centrifuged 20min under the conditions of 12000r/min.
6. detection method as described in claim 1, which is characterized in that in the step (4), substrate solution component is as follows: 2~
5mg/L glutamine, 5~8mg/L6- phosphofructose, 0.7~1mg/L ethylenediamine tetra-acetic acid, 1mL pH value are 7.5
100mmol/L phosphate buffer.
7. detection method as described in claim 1, which is characterized in that in the step (4), substrate solution and GlmS crude enzyme liquid
Volume ratio be 5:1.
8. detection method as described in claim 1, which is characterized in that in the step (4), terminating reaction condition is at 95 DEG C
Manage 4min.
9. detection method as described in claim 1, which is characterized in that in the step (4), centrifugal condition 8000r/min
It is centrifuged 5min.
10. detection method as described in claim 1, which is characterized in that in the step (4), detection glutamic acid yield is to adopt
It is detected with biosensor.
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| CN106929499A (en) * | 2017-04-26 | 2017-07-07 | 扬州日兴生物科技股份有限公司 | A kind of Glucosamine synthase mutant of directional transformation and its application |
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